56

Biochimica et Biophysica Acta, 1023 (1990) 56-62

Elsevier BBAMEM 74775

A monoclonal antibody against pig gastric H + / K + - A T P a s e , which binds to the cytosolic E 1 • K ÷ form Tom J.F. Van U e m

1, Wilbert H.M.

Peters 2 and Jan Joep H . H . M . De Pont 1

J Department of Biochemistry and 2 Division of Gastrointestinal and Liver Diseases, University of Nijmegen, Nijmegen (The Netherlands)

(Received15 September1989)

Key words: ATPase,H+/K+-; Monoclonalantibody; (Hog gastric mucosa)

Monoclonal antibodies were raised against a purified membrane fraction from hog gastric mucosa containing H + / K +-ATPase. The properties of one of these monoclonal antibodies (5B6) were further evaluated. On immunoblot it recognized the 95 kDa peptide of the H+/K+-ATPase-rich membrane fraction. The K+-ATPase activity was inhibited by 65% under standard assay conditions (pH 7.0). At pH 6.0 and 8.0 this enzyme activity was inhibited by 40% and 100%, respectively. The maximal inhibition in inside-out vesicles was also 65% at pH 7.0. The inhibition was uncompetitive with respect to K ÷ and noncompetitive with respect to ATP. Mg2+-ATPase activity and K+-dependent p-nitrophenylphosphatase activity were not influenced. The monoclonal antibody lowered the steady-state phosphorylation level at pH 6.0, 7.0 and 8.0 by 30%, 40% and 60% respectively. The rate of the K +-stimulated dephosphorylation step was not inhibited. These findings demonstrate that 5-B6 recognizes the E a K + dephosphoenzyme at the cytosolic side. °

Introduction The H + / K + - A T P a s e of the gastric mucosa catalyzes the exchange of H ÷ and K ÷ upon hydrolysis of ATP which results in a p H difference of more than 6 between the cytoplasm and gastric lumen [1,2]. The catalytic subunit of this membrane-bound enzyme is related to a number of other cation transport ATPases, including the N a + / K + - A T P a s e and the Ca2+-ATPase [3,4]. These enzymes belong to the class of P-type ATPases as they all have a stable phosphoenzyme intermediate in the enzyme cycle [5,6]. Recently the amino acid sequences of the catalytic subunits of rat and hog gastric H + / K +ATPase have been deduced from the nucleotide sequence of their c D N A [3,7] and the molecular weight of the protein was calculated to be 114000 in either case.

Abbreviations: H+/K+-ATPase (EC 3.6.1.36), magnesium-dependent hydrogen ion transporting and potassium-stimulated adenosine triphosphatase; K+-ATPase, K+-dependent adenosine triphosphatase; Mg2+-ATPase, Mg2+-dependent adenosine triphosphatase; ELISA, enzyme-linkedimmunosorbentassay; PBS, phosphate-bufferedsaline; Tween 20, polyoxyethylenesorbitan monolaurate. Correspondence: J.J.H.H.M. de Pont, Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

There is a 60% homology between the primary sequences of the catalytic subunits of H + / K + - A T P a s e and N a + / K + - A T P a s e [3]. Hydropathy analysis revealed that the H + / K + - A T P a s e subunit probably has eight transmembrane domains as is the case for N a + / K +ATPase and Ca2+-ATPase [3]. Besides these structural similarities, the H + / K + - A T P a s e also has many functional aspects in common with other P-type ATPases. Phosphorylation is on an aspartate residue [4] and hydrolysis of ATP is inhibited by vanadate [8]. The existence of two conformational states, E a and E 2 which have reciprocal affinities for H ÷ and K + at cytosolic and luminal side, respectively, has been proved by limited proteolytic digestion [9] and by fluorescent probes [10,11]. Cycling of the enzyme between these two conformational states, during ATP hydrolysis, gives rise to the translocation of H + to the lumen and of K ÷ to the cytosol. So far relatively little is known about the structure of the enzyme. It is not known, for example, which part of the enzyme is localized at the luminal and which at the cytosolic side; which amino acids contribute to the binding sites for ions and inhibitors as omeprazole [12] and SCH 28080 [13]. Monoclonal antibodies against the H + / K ÷ - A T P a s e could be used as probes to investigate the structure and function of the enzyme. They could clarify whether the gastric enzyme is an oligomer of identical or nonidenti-

0005-2736/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

57 cal 114 kDa subunits [14] and whether it has any immunological similarity to the Na+/K+-ATPase [15]. Monoclonal antibodies against the subunit of the pig gastric H÷/K+-ATPase are already available [16-18]. These monoclonal antibodies have been used for histological studies on H+/K÷-ATPase [16] and to suggest that the C1- channel is part of the function of H + / K +ATPase [18]. In order to get more information about structure and function of H÷/K+-ATPase we prepared monoclonal antibodies against the enzyme. In this paper we describe the characteristics of one of these monoclonal antibodies which inhibits the gastric H+/K÷-ATPase.

NaC1 on three subsequent days. On the fourth day the spleen was removed and spleen cells (5.107 ) were fused with mouse Sp2/0 myeloma cells (2.107) in the presence of 40% (v/v) polyethylene glycol 4000. The cells were washed and cultured in RPMI 1640 medium (Dutch modification, Flow Laboratories, Irvine, Scotland) supplemented with 20% (v/v) fetal calf serum (Flow Laboratories), 2 mM glutamine, 50 /~M 2-mercaptoethanol, 1 mM sodium pyruvate and 50 mg/1 gentamycin. After 3 hours a solution containing hypoxanthine, aminopterin and thymidine (final concentrations 0.1 mM, 0.4 #M and 16/~M) was added to the medium and cells were plated out.

Materials and Methods

Production and partial purification of monoclonal antibodies

Preparation of gastric H + / K +-ATPase

Hybridomas producing antibodies were cloned by limited dilution and injected (5.106 cells) intraperitoneally into BALB/c mice primed with pristane (0.5 ml i.p.) 2-4 weeks before. After 10 days antibody-rich ascites fluid was obtained every 2-3 days and centrifuged for 10 min at 2000 × g to remove cell debris. The supernatant was precipitated with ammonium sulfate (pH 7.4, adjusted with Tris) at 50% saturation. The 12000 × g precipitate was resuspended in a volume of 20 mM Tris-HC1 (pH 7.4) equal to the original volume of the ascites, dialyzed against 100 volumes of 20 mM Tris-HC1 (pH 7.4) at 4 ° C with three changes of the dialysate and stored at - 2 0 o C. The subclasses of the monoclonal antibodies were determined with subclassspecific antibodies according to Ouchterlony [20].

Isolation of the H +/K+-ATPase has been carried out according to the procedure previously reported by Skrabanja et al. [19] with some modifications. Stomachs of freshly slaughtered pigs were transported to the laboratory on ice. After flushing with tap water the fundic region was placed during 30 min in homogenization buffer containing 250 mM sucrose and 20 mM Tris-HC1 (pH 7.4). Mucus was removed by wiping the tissue with paper towels and the mucosa was scraped off from the underlying muscular layer. The scraped material was placed in homogenization buffer and homogenized with a Braun teflon-glass homogenizer by three up-down strokes of the rotating pestle (250 rev./min). The homogenate was centrifuged for 30 min at 20 000 × g. The resulting supernatant was centrifuged for 60 min at 100 000 × g. The pellet was resuspended in homogenization buffer and centrifuged on top of a discontinuous gradient of 7% (w/v) Ficoll-250 mM sucrose in 20 mM Tris-HC1 (pH 7.4) over 37% (w/v) sucrose in 20 mM Tris-HC1 (pH 7.4). After 60 min centrifugation at 150000 × g the 250 mM sucrose/7% (w/v) Ficoll-250 mM sucrose interface containing the vesicular H+/K+-ATPase fraction and the 7% (w/v) Ficoll-250 mM sucrose/37% (w/v) sucrose interface containing the open membrane H+/K+-ATPase fraction were diluted in 20 mM Tris-HC1 (pH 7.4) and centrifuged for 60 min at 150 000 × g. The final pellets were resuspended in 20 mM Tris-HC1 (pH 7.4) and frozen at - 20 o C.

Hybridoma production The antigen was H+/K+-ATPase purified from hog stomach and emulsified in complete Freund's adjuvant. Several 6-week-old female BALB/c mice were immunized intraperitoneally with 200/tg of enzyme. After 3 weeks, sera were drawn for evaluation of titer against gastric H+/K+-ATPase. One mouse was selected on the basis of the highest titer and boostered by tail vein injection of 100 #g antigen in 0.3 ml of 0.9% (w/v)

ELISA Reagents per well and incubation times were as follows: 0.5/tg of gastric H+/K+-ATPase in 50 #1 PBS was immobilized by overnight evaporation in wells of flat-bottomed, 96-well microtiter plates (Costar, Cambridge, MA, U.S.A.). Blocking was done by incubating with a solution of 1% (w/v) gelatin in PBS for 2 h at room temperature whereafter the plates were washed with PBS containing 0.05% (v/v) Tween 20. Incubation for 1 h at room temperature with antibodies from hybridoma culture media was followed by washing with PBS/Tween and subsequent incubation with peroxidase conjugated rabbit-anti-mouse immunoglobulins (Dakopatts, Denmark) diluted 1 : 800 in PBS with 1% gelatin. After washing a substrate solution containing 0.8 mg/ml 5-aminosalicylic acid in 50 mM phosphate buffer (pH 6.0) and 0.024% (w/v) hydrogen peroxide was added. Absorption at 492 nm was measured after 30 min using a Titertek Multiscan plate reader (Flow Laboratories).

Gel electrophoresis and immunoblotting Purified H+/K+-ATPase was electrophoresed on 10% (w/v) SDS-polyacrylamide slab gel according to Laemmli [21]. The separated proteins were transferred to

58 nitrocellulose by electroblotting [22]. The blots were incubated first for 2 h with PBS-I% (w/v) gelatin-0.05% (w/v) Tween 20 (Buffer I) to block nonspecific reactive sites, then for 16 h with 1 : 100 dilutions of monoclonal antibody 5-B6 in Buffer I. After washing five times in PBS-0.05% (w/v) Tween 20 (Buffer II) the blots were incubated for 1 h with 1:250 dilutions of rabbit-antimouse immunoglobulin (Dakopatts, Denmark) in Buffer I, washed again with Buffer II and incubated for another hour with 1 : 250 dilution of mouse peroxidase-antiperoxidase immunoglobulin (Dakopatts, Denmark) in Buffer I. Finally the blots were washed with Buffer II and substrate solution (60 mg 4-chloronaphthol, 60/~1 30% (w/v) hydrogen peroxide, 20 ml methanol, 100 ml PBS) was added.

A TPase and p-nitrophenylphosphatase assay K+-ATPase activity and K+-p-nitrophenylphosphatase activity of the enzyme were determined according to Schrijen et al. [23]. Gastric H+/K+-ATPase, 100 /~1 with a concentration of 60 #g protein/ml, was preincubated for 10 min at 37 °C with 100 /zl of a diluted monoclonal antibody solution with concentrations as indicated in the figures. Dilutions of enzyme and monoclonal antibody were made in 30 mM imidazole-HC1 (pH 7.0). The reaction was started by adding 200 ~tl medium containing 10 mM Na2ATP or 10 mM pnitrophenyl phosphate, 10 mM Mg 2+, 0.2 mM ouabain, 30 mM imidazole-HC1 (pH 7.0) and 40 mM KC1 or 40 mM choline chloride. Stopping of the reaction and further treatment of the samples were carried out as described before [24]. The K+-ATPase and K+-p nitrophenylphosphatase activities were obtained from the difference in activity with KC1 and choline chloride, respectively. Protein was determined according to Lowry et al. [25], using bovine serum albumin as standard. Radioactive A TPase assay Aliquots of 50/~1 enzyme (0.08 mg/ml) were preincubated for 10 min at 37°C with 50 /~1 monoclonal antibody solution at various concentrations and 50 /~1 medium containing 60 mM imidazole-HC1 (pH 7.0), 0.4 mM ouabain and 80 mM KC1 or 80 mM choline chloride. The reaction was initiated by adding 50 ~tl of a solution containing [3'-32p]ATP and Mg z+ at a ratio of 0.5 and at various concentrations. The reaction was stopped by adding 800 /xl 5% (w/v) trichloroacetic acid/10% (w/v) Norit. After centrifugation for 10 min at 1500 x g (Heraeus Christ) 200/~1 of the supernatant was counted in a Packard 2200CA liquid scintillation analyzer. Enzyme phosphorylation and dephosphorylation The phosphorylation and dephosphorylation reactions were carried out as described by Helmich-de Jong et al. [26]. Aliquots of 50 #1 H+/K+-ATPase (0.3

mg/ml) were preincubated for 10 min at room temperature with 50/~1 monoclonal antibody solution at various concentrations and 4 mM MgC12- The phosphorylation reaction was started by adding 100 #1 10 ~M [3'32p]ATP. After 10 s at 20°C the reaction was stopped by adding 5 ml 5% (w/v) trichloroacetic acid/0.1 M H3PO4. The effect of the monoclonal antibody on the rate of the potassium stimulated dephosphorylation was studied at 0-4 ° C. Phosphorylation was first initiated by adding 50-/d aliquots of 10 #M [3'-32p]ATP, 10/~M MgC12 to 50/~1 aliquots of H+/K+-ATPase (0.2 mg/ml). After 10 s dephosphorylation was started by adding ATP (final concentration 1 mM), KC1 at various concentrations and monoclonal antibody. The reaction was stopped by adding 5 ml 5% (w/v) trichloroacetic acid/0.1 M H3PO 4. Further processing was carried out as described by Schuurmans Stekhoven et al. [27]. Results

Preparation of monoclonal antibodies By ELISA screening of the produced hybridomas six monoclonal antibodies were obtained which showed a positive reaction against the purified hog gastric H + / K +-ATPase. Four of them inhibited the K +-ATPase activity. One of them, called 5-B6, gave the strongest inhibition and was further characterized. Double immuno-diffusion with Ig subclass specific anti-mouse immunoglobulins showed that 5-B6 is of subclass IgG 1. By Western blot analysis of the purified H+/K+-ATPase membrane fraction, 5-B6 recognized a 95 kDa peptide (Fig. 1). There was no cross reactivity with rabbit kidney Na÷/K+-ATPase. Effect on H + / K +-A TPase The gastric H+/K+-ATPase preparation contains K+-ATPase, K+-p-nitrophenylphosphatase as well as a MgZ+-ATPase activity. The effect of monoclonal antibody 5-B6 on these enzyme activities was studied with a non-inhibitory monoclonal antibody, directed against H+/K+-ATPase and also partially purified, as control. Fig. 2 shows the inhibition of K+-ATPase activity with various amounts of 5-B6 (0.05 to 150 #g/ml) and with three different H+/K+-ATPase concentrations (5, 15 and 50 #g/ml) in the reaction mixture. The maximal inhibition obtained was 65% for all three H + / K +ATPase concentrations used. Variation of the preincubation time had no effect on the degree of inhibition. Activities of K+-p-nitrophenylphosphatase and Mg 2+ATPase were not influenced (data not shown). The maximal extent of inhibition of K+-ATPase activity by 5-B6 varied with the pH in the reaction mixture (Fig. 3). At pH 6.0, 7.0 and 8.0 the maximal inhibition of the K+-ATPase activity was 40%, 65% and 100%, respectively. The half-maximal inhibitory con-

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Fig. 3. Inhibition of K+-ATPase activity of H+/K+-ATPase by monoclonal antibody 5-B6 as a function of pH. 15 btg/ml leaky H+/K+-ATPase vesicles were preincubated with different concentrations of monoclonal antibody 5-B6 in 30 mM imidazole-HCl at pH 6.0 (©), pH 7.0 (zx) or pH 8.0 (D) at 37°C for 10 min. K+-ATPase activity was determined as described in Materials and Methods (representative of three experiments). The 100% activity at pH 6.0, 7.0 and 8.0 was 29.8, 79.8 and 19.3 itmol Pi per mg per h, respectively.

Fig. 1. Western blot of pig gastric H+/K+-ATPase stained with monoclonal antibody 5-B6. (A) Amido Black staining of proteins on nitrocellulose after electroblotting of a 10% SDS polyacrylamide gel. Lane 1, 5 /zg gastric microsomal membrane proteins. Lane 2, 5 /.tg purified H+/K+-ATPase, Molecular mass markers are 94, 67, 43, 30 and 20.1 kDa. (B) Western blot stained with the anti-H+/K+-ATPase monoclonal antibody 5-B6. Lane 1, 3 /~g gastric microsomal membrane proteins. Lane 2, 1 # g purified H +/K +-ATPase.

centration of the antibody did not change with p H and was 15 #g of a n t i b o d y / m l when 15 # g / m l H + / K +ATPase was used. The inhibition of the K+-ATPase activity by 5-B6 was measured as function of both K + as well as ATP concentration. Fig. 4 shows that 2 0 / ~ g / m l 5-B6 in an assay medium containing 15 /~g/ml H + / K + - A T P a s e lowered the Vma~ value from 80 to 35/~mol Pi per mg per h. The K m value for K + was lowered from 4.8 to 1.9 mM. These results indicate that the interaction between K + and 5-B6 is uncompetitive. Variation of the A T P concentration at a fixed K + concentration (Fig. 5) showed that 20 btg/ml 5-B6 in an assay medium con-

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Fig. 5. Effect of ATP concentrations on the inhibition of K-ATPase activity of H+/K+-ATPase by monoclonal antibody 5-B6. 15/zg/ml H+/K+-ATPase were preincubated with 20/~g/ml 5-B6 at 37 °C for 10 min. K+-ATPase activity was determined as described in Materials and Methods with assay media containing 20 mM KC1, 0.1 mM ouabain, 30 mM imidazole-HC1(pH 7.0), [T-3zP]ATPconcentrations varying between 0.025 and 0.5 mM and MgC12concentrations varying between 0.05 and 1 raM, thereby maintaining an ATP/Mg 2+ ratio of 1 : 2. O, Control; o, 5-B6 (representative of three experiments).

t a i n i n g 15 /~g/ml H + / K + - A T P a s e decreased the Vm~x value from 100 to 25 /~mol Pi per mg per h. The K m values did n o t change. The effect of 5-B6 o n the K ÷A T P a s e reaction with respect to A T P was therefore noncompetitive. Effects on p h o s p h o e n z y m e The effect of m o n o c l o n a l a n t i b o d y 5-B6 o n the steady-state level of p h o s p h o e n z y m e i n the absence of K ÷ a n d at p H 6.0, 7.0 a n d 8.0, respectively, is shown i n Fig. 6. The steady-state p h o s p h o r y l a t i o n level at p H 6.0,

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Fig. 7. Effect of monoclonal antibody 5-B6 on K+-stimulated dephosphorylation of H+/K+-ATPase. Phosphorylation at 0°C of 100 /~g/ml H+/K+-ATPase was carried out as described in Materials and Methods. After 10 s dephosphorylation of the phosphoenzyme was started with addition of cold ATP (final concentration 1 mM), 5-B6 (final concentration 100 /~g/ml) and KC1 to a final concentration of 0.1 mM (m) or 1 mM (4). Blank symbols represent control values. Stopping of the phosphorylation reaction and determination of phosphoenzyme levels were carried out as described in Materials and Methods. The phosphoenzyme levels, in the absence of 5-B6 (100% values), were 0.92 nmol/mg protein (representative of three experiments).

7.0 a n d 8.0 was c o n c e n t r a t i o n d e p e n d e n t l y reduced to a m a x i m a l extent of 30%, 40% a n d 60%, respectively. The h a l f - m a x i m a l i n h i b i t o r y c o n c e n t r a t i o n of 5-B6 i n all cases was 30 / ~ g / m l with 75 / ~ g / m l H ÷ / K + - A T P a s e used. W h e n the effect of 5-B6 o n the steady-state phosphor y l a t i o n level was studied without p r e i n c u b a t i n g with 5-B6 (the m o n o c l o n a l a n t i b o d y was a d d e d at the same time as p h o s p h o r y l a t i o n was started) the same extent of i n h i b i t i o n was seen (data n o t shown). These results i n d i c a t e d a very r a p i d b i n d i n g of m o n o c l o n a l a n t i b o d y to gastric H + / K + - A T P a s e .

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Fig. 6. Effect of monoclonal antibody 5-B6 on steady-state phosphoenzyme levels of H+/K+-ATPase at different pH. 75 /tg/rnl purified H +/K+-ATPase were preincubated in 30 mM imidazole-HC1 pH 6.0 (o), pH 7.0 (zx)and pH 8.0 (ra) with various concentrations of 5-B6 and 4 mM MgC12. Initiation of the phosphorylation and determination of phosphoenzyme levels were carried out as described in Materials and Methods. The phosphoenzyme levels, in the absence of 5-B6 (100%), were 0.75 nmol/mg protein (representative of three experiments).

TABLE l Inhibition of K +-.4TPase activity by 5-B6 in inside-out and in leaky vesicles

80 /tg/ml H+/K+-ATPase (specific activity 85 /~mol Pi/mg per h) was preincubated for 15 min at room temperature with 10 /xM nigericin (inside-out vesicles) or with 0.3% (w/v) saponin (leaky vesicles). The K+-ATPase reaction was started by adding 100 /tl enzyme solution to 300 #l isotonic assay medium with a final concentration of 100 /xg/rrd 5-B6. The values are given as percentages relative to K+-ATPase activity with control mouse IgG. Each value is the average of three separate determinations, each of which is done in duplicate. Percentage residual K +-ATPase activity 1. Inside-out vesicles 2. Leaky vesicles

34 + 7 28 + 4

61 The effect of 5-B6 on the K+-stimulated dephosphorylation rate is shown in Fig. 7. In this experiment dephosphorylation was initiated after 10 s by adding cold ATP (final concentration 1 mM) along with the indicated concentrations of KC1 and 5-B6. The monoclonal antibody did not affect the rate of the K+-stimu lated dephosphorylation, nor the second phase in the dephosphorylation, which is interpreted as interconversion from E] • P to E 2 • P [28].

Sidedness of inhibition of gastric H+/K+-ATPase by 5-B6 For determination of the sidedness of binding of monoclonal antibody 5-B6 on gastric H + / K ÷ - A T P a s e freshly isolated inside-out vesicles were used. Table I shows that the maximal level of inhibition of K ÷-ATPase activity was identical in inside-out vesicles as well as in leaky H + / K + - A T P a s e vesicles. These results indicate that 5-B6 binds to the cytosolic side of gastric H + / K +ATPase. Discussion In the present study, the interactions of monoclonal antibody 5-B6 with H + / K + - A T P a s e have been investigated. To clarify interpretation of the interactions of 5-B6 with H + / K + - A T P a s e the following enzymatic mechanism of gastric H + / K + - A T P a s e has to be considered (Fig. 8) [28-30]. In this mechanism, which requires several steps related to binding of substrate and cationic ligands, E 1 and E 2 represent conformational states of the enzyme with cation binding sites facing the cytosolic and luminal side, respectively. When considering the phosphorylation reaction, the E~ conformation can interact competitively with either H ÷ or K +. The E l . K ÷ form relatively slowly reacts with ATP, forming an E 1 • A T P . K ÷ complex. At higher H ÷ concentrations K ÷ may also be displaced from the

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Fig. 8. A reactioncycle of H+/K+-ATPase. E] and E2 represent conformational states of the enzyme with cation binding sites facing the cytosol and lumen, respectively. I represents the inhibitory monoclonal antibody 5-B6.

enzyme and E l . H ÷ is formed. This form reacts with ATP and the E 1 • A T P . K ÷ with H ÷ to a E 1 • A T P . H ÷ complex which is subsequently phosphorylated (step 1). The E l - P form then generated, is ADP-sensitive [26] and converts spontaneously to E2P with concomitant transport of H ÷ (step 2) [31]. This form has a high affinity for K ÷ at the luminal side and is rapidly hydrolyzed (step 3) [28]. The E 2 • K ÷ form then converts to the E~. K + form with consequent countertransport of K ÷ (step 4) [32]. The monoclonal antibody 5-B6, which binds to the 95 kDa peptide in the purified H + / K + - A T P a s e fraction, inhibited the K+-ATPase activity in a concentration dependent way. The maximal extent of inhibition was p H dependent and was maximal at p H 8.0. Lineweaver-Burk plots showed that inhibition of the K ÷ATPase by 5-B6 at pH 7.0 was noncompetitive with respect to ATP and uncompetitive with respect to K ÷. Since the stimulation of the ATP hydrolysis by K ÷ is mediated through an increase of the dephosphorylation rate of E 2 • P, these findings would suggest an interaction of 5-B6 with the E 2- K + or the E 1- K ÷ form. A distinction between these two dephospho-forms can not be made since the E 2 • K ÷ to E] • K ÷ conversion is not a rate-limiting step in the enzyme cycle at the high A T P / K ÷ ratio used [28]. The finding however, that 5-B6 recognizes an epitope on the cytosolic side, would favour binding of 5-B6 to the E] • K ÷ form. The varying degree of maximal inhibition of the K+-ATPase activity at different p H can be explained by the influence of [H ÷] on step 1 and step 3 of the enzyme cycle [29-30]. An alkaline p H increases the rate of the K+-stimulated dephosphorylation and stabilizes the E l - K ÷ form of the enzyme which has low affinity for ATP [29]. This results in an accumulation of the E 1 • K ÷ form during ATP hydrolysis and therefore increases the maximal extent of inhibition. An acidic p H on the other hand, stabilizes the acid-stable phosphoenzyme by reducing the affinity for luminal K ÷ and increases the rate and extent of phosphorylation. As a consequence, the maximal extent of inhibition by 5-B6 decreases. The finding that the K+-p-nitrophenylphosphatase activity is not inhibited by 5-B6 is consistent with binding of 5-B6 to the E 1 • K ÷ form, since hydrolysis of p-nitrophenylphosphate requires a dephosphoenzyme with a cytosolic K ÷ stimulation site (El • K ÷) [30] or a luminal K ÷ stimulation site (E 2 • K ÷) [33]. An alternative explanation might be that 5-B6 binds to a form of the enzyme which is intermediate between E 1 • K ÷ and E 2 • K ÷. This form E(K ÷) would represent an enzyme form which occludes K ÷. Such a form has not yet been demonstrated for H + / K + - A T P a s e in contrast to N a + / K + - A T P a s e [34,35] where such a form easily can be found. The steady-state phosphorylation level of H + / K +ATPase was reduced by 5-B6 in a concentration depen-

62 dent way. T h e m a x i m a l extent of reduction was also p H dependent. A reduction of the steady-state level of p h o s p h o e n z y m e does n o t indicate whether i n h i b i t i o n of formation, or s t i m u l a t i o n of b r e a k d o w n has occurred. Since 5-B6 neither accelerated the s p o n t a n e o u s (data n o t shown) a n d the K + - s t i m u l a t e d d e p h o s p h o r y l a t i o n n o r influenced the interconversion from E 1 • P to E2-P, it seems possible that b i n d i n g of 5-B6 prevents the p h o s p h o e n z y m e formation. Normally, the steady-state level of p h o s p h o e n z y m e does n o t change with pH, as does the rate of p h o s p h o r y l a t i o n in the absence of K ÷ [29]. I n the case of reduction of the EP level b y 5-B6 however, an alkaline p H might cause a n i n h i b i t i o n of the p h o s p h o r y l a t i o n rate b y 5-B6 through stabilizing a n enzyme form which inhibits or slows d o w n p h o s p h o rylation. This is p r o b a b l y an E 1 form w i t h o u t H ÷ b o u n d to it. A n acidic p H o n the other h a n d w o u l d favour the E 1 . H ÷ form, a n d as a result a smaller m a x i m a l extent of reduction of EP level b y 5-B6 w o u l d be reached. The difference i n the m a x i m a l extent of i n h i b i t i o n of the K + - A T P a s e activity, a n d of r e d u c t i o n of the steady-state p h o s p h o e n z y m e level at each p H value, can be explained b y the absence of the E 1 • K + form d u r i n g p h o s p h o r y l a t i o n a n d for which 5-B6 has a greater affinity t h a n for the E 1 form w i t h o u t H + b o u n d to it. Therefore the enzyme c o n f o r m a t i o n differs i n the E 1 • K + or E~ a n d E 1 • H ÷ in such a way that effective b i n d i n g with m o n o c l o n a l a n t i b o d y 5-B6 is selective for the E 1 • K ÷ form.

References 1 Sachs, G., Chang, H.H., Rabon, E., Schackman, R., Lewin, M. and Saccomani, G. (1976) J. Biol. Chem. 251, 7690-7698. 2 Ganser, A.L. and Forte, J.G. (1973) Biochim. Biophys. Acta 307, 169-180. 3 Shull, G.E. and Lingrel, J.B. (1986) J. Biol. Chem. 261, 16788-16791. 4 Walderhaug, M.O., Post, R.L., Saccomani, G., Leonard, R.T. and Briskin, D.P. (1985) J. Biol. Chem. 260, 3852-3859. 5 Pedersen, P.L. and Carafoli, E. (1987) Trends Biochem. Sci. 12, 146-151. 6 Pedersen, P.L. and Carafoli, E. (1987) Trends Biochem. Sci. 12, 186-189. 7 Maeda, M., Ishizaki, J. and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157, 203-209. 8 Failer, L.D., Rabon, E. and Sachs, G. (1983) Biochemistry 22, 4676-4685. 9 Helmich-de Jong, M.L., Van Ernst-de Vries, S.E. and De Pont, J.J.H.H.M. (1987) Bioehim. Biophys. Acta 905, 358-370. 10 Helmich-de Jong, M.L., Van Duynhoven, J.P.M., Schuurmans

Stekhoven, F.M.A.H. and De Pont, J.J.H.H.M. (1986) Biochim. Biophys. Acta 858, 254-262. 11 Jackson, R.G., Mendlein, J. and Sachs, G. (1983) Biochim. Biophys. Acta 731, 9-15. 12 Lorentzon, P., Jackson, R., Wallmark, B. and Sachs, G. (1987) Biochim. Biophys. Acta 897, 41-51. 13 Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, J., Rabon, E. and Sachs, G. (1987) J. Biol. Chem. 262, 2077-2084. 14 Faller, L.D., Smolka, A. and Sachs, G. (1985) in The Enzymes of Biological Membranes, 2nd Edn., Vol. 3 (Martonosi, A.N., ed.), pp. 431-438. 15 Peters, W.H.M., Ederveen, A.G.H., Salden, M.H.L., De Pont, J.J.H.H.M. and Bonting, S.L. (1984) J. Bioenerg. Biomembr. 16, 223-232. 16 Smolka, A., Helander, H.F. and Sachs, G. (1983) Am. J. Physiol. 245, G589-G596. 17 Smolka, A. and Weinstein, W. (1986) Gastroenterology 90, 532-539. 18 Asano, S., Inoie, M. and Takeguchi, N. (1987) J. Biol. Chem. 262, 13263-13268. 19 Skrabanja, A.T.P., Van der Hijden, H.T.W.M. and De Pont, J.J.H.H.M. (1987) Biochim. Biophys. Acta 903, 434-440. 20 Ouchterlony, O. (1959) Lancet 1,346-348. 21 Laernrnli, U.K. (1970) Nature 227, 680-685. 22 Burnette, W.N. (1981) Anal. Biochem. 112, 195-203. 23 Schrijen, J.J., Luyben, W.A.H.M., De Pont, J.J.H.H.M. and Bonting, S.L. (1980) Biochim. Biophys. Acta 597, 331-344. 24 Schoot, B.M., Schoots, A.F.M., De Pont, J.J.H.H.M., Schuurmans Stekhoven, F.M.A.H. and Bonting, S.L. (1977) Biochim. Biophys. Acta 483, 181-192. 25 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 26 Helmich-de Jong, M.L., Van Ernst-de Vries, S.E., De Pont, J.J.H.H.M., Schuurmans Stekhoven, F.M.A.H. and Bonting, S.L. (1985) Biochim. Biophys. Acta 821, 377-383. 27 Schuurmans Stekhoven, F.M.A.H., Swarts, H.G.P., De Pont, J.J.H.H.M. and Bonting, S.L. (1980) Biochim. Biophys. Acta 597, 100-111. 28 Wallmark, B., Stewart, H.B., Rabon, E., Saccomani, G. and Sachs, G. (1980) J. Biol. Chem. 255, 5313-5319. 29 Stewart, H.B., Wallmark, B. and Sachs, G. (1981) J. Biol. Chem. 256, 2682-2690. 30 Ljungstr~m, M., Vega, F.V. and MArdh, S. (1984) Biochim. Biophys. Acta 769, 220-230. 31 Fendler, K., Van der Hijden, H.T.W.M., Nagel, G., De Pont, J.J.H.H.M. and Bamberg, E. (1988) in The Na+,K+-pump (Skou, J.C., Norby, J.G., Maunsbach, A.B. and Esmann, M., eds.), Part A, pp. 501-510, A.R. Liss, New York. 32 Schackman, R., Schwartz, A., Saccomani, G. and Sachs, G. (1977) J. Membr. Biol. 32, 361-381. 33 Saccomani, G., Barcellona, M.L. and Sachs, G. (1981) J. Biol. Chem. 256, 12405-12410. 34 Beaug6, L.A. and Glynn, I.M. (1979) Nature 280, 510-512. 35 Glynn, I.M., Howland, J.L. and Richards, D.E. (1985) J. Physiol. London 368, 453-469.

K(+)-ATPase, which binds to the cytosolic E1.K+ form.

Monoclonal antibodies were raised against a purified membrane fraction from hog gastric mucosa containing H+/K(+)-ATPase. The properties of one of the...
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